1. Field of the Invention
The present invention relates to biomedical implants and devices, tissue engineering and regenerative medicine, and health care products. More particularly, embodiments of the present invention relate to systems and methods for production and control of 3-D architecture and morphology of nano-cellulose biomaterials produced by bacteria using novel biofabrication processes, such as 3-D Bioprinting. Representative processes according to the invention involve control of the rate of production of biomaterial by bacteria achieved by meticulous control of addition of fermentation media using a microfluidic system. If desired, porosity and interconnectivity of pores within the resultant 3-D architecture can be achieved by porogen introduction using, for example, ink-jet printer technology.
2. Description of the Related Art
Tissue engineering and regenerative medicine provide the revolutionary solution for tissue and organ replacement. Millions of patients suffer today from lack of organ due to trauma or loss of organ due to disease such as cancer followed by surgery. Increased elder population is of need of worn tissue and organs in order to be able to live a comfortable life. Synthetic materials have only had limited success as implants due to a lack of biocompatibility and mismatch of biomechanical performance. Natural polymers are much better suited as biomaterials due to generally better biocompatibility and improved tissue integration. Tissue engineering is based on the use of scaffold materials with very well defined morphology to host the cells and provide support for cell producing extracellular matrix. The major bottle-neck in the field of tissue engineering and regenerative medicine is a lack of scaffolds with tailor-made architecture which is caused by lack of production technology to control biomaterial's architecture in great detail.
Natural polymers such as collagen or elastin have been extensively evaluated as implants and scaffold for tissue engineering. The major drawback of these protein based materials is immunological response and difficulty to process these materials into porous scaffolds with predetermined morphology. Polysaccharides such as hyaluronic acid, alginates, chitosan and cellulose have been recently successfully introduced as scaffolds for tissue engineering. They can be easily sterilized and they do not cause immunological response. Cellulose (β-1→4-glucan) is a natural polysaccharide biosynthesized by a majority of plants. Cellulose is attractive as a biomaterial because of its good mechanical properties (Bäckdahl, H., Helenius, G., Bodin, A., Johansson, B., Nanmark, U., Risberg, B., and Gatenholm, P., Bacterial Cellulose as Potential Scaffold for Tissue Engineered Blood Vessels: Mechanical Properties and Cell Interactions, Biomaterials, 27, 2141-2149 (2006)), hydroexpansivity (Gelin, K., Bodin, A., Gatenholm, P., Mihranyan, A., Edwards, K and Strømme, M., Characterization of Water in Bacterial Cellulose Using Dielectric Spectroscopy and Electron Microscopy, Polymer, 48, 7623-7631 (2007)), biocompatibility (Gatenholm, P., and Klemm, D., Bacterial Nanocellulose as a Renewable Material for Biomedical Applications, MRS Bulletin, 35 (3), 208-213, 2010), and structural stability within a wide range of temperatures and pH levels. In addition to being synthesized in vast amounts as a structural material in the walls of plants, cellulose can also be produced as an exopolysaccharide, i.e. biosynthetic cellulose (BC) synthesized by bacteria such as Acetobacter xylinum (Gatenholm, P., and Klemm, D., Bacterial Nanocellulose as a Renewable Material for Biomedical Applications, MRS Bulletin, 35 (3), 208-213, (2010)).
BC has been recently evaluated in several biomedical applications. In addition to being used for microsurgery (Gatenholm, P., and Klemm, D., Bacterial Nanocellulose as a Renewable Material for Biomedical Applications, MRS Bulletin, 35 (3), 208-213, (2010)), it has been evaluated as vascular grafts (Bäckdahl, H., Risberg, B., and Gatenholm, P., Observation on Bacterial Cellulose Tube Formation for Application as Vascular Graft, Materials, Science and Engineering, Part C in press 2010), cartilage replacement (Svensson, A., Nicklasson, E. Harrah, T., Panilaitis, B., Kaplan, D. Brittberg. M, and Gatenholm, P., Bacterial Cellulose as a Potential Scaffold for Tissue Engineering of Cartilage, Biomaterials, 26, 419-431 (2005)), bone grafts (Zaborowska, M., Bodin, A., Bäckdahl, H., Popp, J., Goldstein, A., and Gatenholm, P., Microporous Bacterial Cellulose as a Potential Scaffold for Bone Regeneration, Acta Biomaterialia, 6 (7), 2540-2547, 2010), and meniscus implant (Bodin, A., Concaro, S., Brittberg, M., and Gatenholm, P., Bacterial Cellulose as a Potential Meniscus Implant, Journal of Tissue Engineering and Regenerative Medicine, 48, 7623-7631 (2007)).
The high water content of biosynthetic cellulose, around 99%, makes it attractive to be used as a hydrogel, which is known for its favorable biocompatible properties and low protein adsorption. BC is a versatile material that can be manufactured in various sizes and shapes. The process of manufacturing is biotechnological process and requires detailed control of bacterial proliferation, migration and rate of production of cellulose. In a typical static culture there is, however, only very limited control of nano-cellulose production process.
Another of the challenges of using biosynthetic cellulose as a scaffold for tissue engineering has been its relatively tight structure of network of cellulose nanofibrils. It has been shown that chondrocytes cells have rather colonized surface of the material than migrating into the network (Svensson, A., Nicklasson, E. Harrah,T., Panilaitis, B., Kaplan, D. Brittberg. M, and Gatenholm, P., Bacterial Cellulose as a Potential Scaffold for Tissue Engineering of Cartilage, Biomaterials, 26, 419-431 (2005)). Attempts have been made to promote the migration of smooth muscle cells (SMC) into the BC network by using chemical attractants. SMC were, however only able to enter 10 μm. Recently, a new technology using porogens has been developed to create macroporosity in BC structure (Backdahl, H., Esguerra, M., Delbro, D., Risberg, B., and Gatenholm, P., Engineering microporosity in bacterial cellulose scaffolds, Journal of Tissue Engineering and Regenerative Medicine, 2 (6), 320-330 (2008)).
This technique resulted in the preparation of macroporous structures which was found to support smooth muscle cells (Backdahl, H., Esguerra, M., Delbro, D., Risberg, B., and Gatenholm, P., Engineering microporosity in bacterial cellulose scaffolds, Journal of Tissue Engineering and Regenerative Medicine, 2 (6), 320-330 (2008)). The process of preparation of macroporous BC is however based on several steps which include preparation of wax porogen particles, fusing particles and fermentation process with porogens. Using this process, only thin macroporous membranes have been produced. Although the macroporous BC showed promising properties, the manufacturing process is not possible to use for mass production.
The synthesis of nano-cellulose by bacteria such as Acetobacter xylinum is taking place between the outer and cytoplasma membrane. Cellulose is a product of carbon metabolism and, depending on the physiological state of the cell, involves either the pentose phosphate cycle or the Krebs cycle coupled with glucogenesis. The growing glucan chains aggregate and are exported through catalytic sites that are linearly arranged on each cell. Bacteria assemble glucan chains into microfibrils and subsequently into a ribbon configuration. In the normal static conditions bacteria will form a pellicle (flake) at the surface of the culture medium. This pellicle will grow in thickness slightly but the thickness is limited by the supply of oxygen and nutrients to the bacteria. Shaped cellulose with limited thickness has been produced in the method using a tubular bioreactor as described in WO2001061026. The oxygen delivery through the silicon support has been explored for manufacturing of tubes for applications such as vascular grafts, as described in EP2079845 and WO2008040729 A2. These structures were however very thin (less than 3 mm thick).
Fermentation conditions play a major role in the determination of material properties of Biosynthetic Cellulose. Use of fermentors and bioreactors has only very limited success due to bacteria sensitivity to agitation and their ability to switch off cellulose production.
In conclusion, the successful use of Biosynthetic Cellulose as implant and scaffold for tissue engineering and regenerative medicine requires a new innovative biofabrication process by which the 3-D shape larger and robust structures, morphology, biomechanical properties and porosity could be controlled in great detail. Such process is described in this patent application and is called 3-D Bioprinting.